Methods for inducing a natural killer (NK) cell-mediated immune response and for increasing NK cell activity

ABSTRACT

This disclosure relates to methods of inducing a natural killer (NK) cell-mediated immune response and increasing NK activity in a mammal for the treatment of tumors and virus infections, comprising isolating peripheral blood mononuclear cells (PBMCs) from a subject, exposing them in vitro to a protein conjugate comprising granulocyte macrophage colony stimulating factor (GM-CSF) covalently linked to a soluble peptide antigen to activate the PBMCs, and administering the activated PBMCs to the subject. The method also relates to assessing NK cell activity of activated PBMCs to determine whether the subject has had a therapeutically effective response to the protein conjugate.

This patent application is a continuation of U.S. patent applicationSer. No. 12/077,823, filed Mar. 21, 2008, now granted as U.S. Pat. No.8,153,120, which claims priority to U.S. Provisional Patent ApplicationNo. 60/896,461 filed on Mar. 22, 2007, which is incorporated herein inits entirety by reference.

REFERENCE TO SEQUENCE LISTING, TABLE OR COMPUTER PROGRAM

A Sequence Listing is being submitted with this application in the formof a text file, created 21 Mar. 2012, and named“576368130US01SeqList.txt” (24,576 bytes), the contents of which areincorporated herein by reference in their entirety.

FIELD OF THE INVENTION

The present invention relates to the fields of biology and immunology.More particularly, it relates to methods for inducing a natural killer(NK) cell-mediated immune response and increasing the NK cell activityof a mammal in order, for example, to treat tumors or viral infections.

BACKGROUND OF THE INVENTION

The immune system is comprised of many different cell types, factors andorgans. These include lymphocytes, monocytes and polymorphonuclearleukocytes, numerous soluble chemical mediators (cytokines and growthfactors), the thymus, postnatal bone marrow, lymph nodes, liver andspleen. All of these components work together through a complexcommunication system to fight against microbial invaders such asbacteria, viruses, fungi and parasites, and against newly arisingmalignant (tumor) cells. NK cells are bone marrow-derived lymphocytes ofthe innate arm of the immune system. They are phenotypically defined asexpressing the low affinity receptor for the Fc protein of IgG(FcRγIIIA, CD16) and CD 56 in the absence of T cell receptor and itsassociated CD3 complex (Perussia et al., 2005, Molecular Immunology 42:385-395).

NK cells have vital importance as a first line of defense againstinfection and tumor proliferation while the adaptive immune system isbeing activated (French et al., 2003, Current Opinion in Immunology 15:45-51). The primary role of NK cells is to eliminate infected orcancerous cells by direct cellular cytotoxicity (Van der Broek et al.,2000, Eur. J. Immunology 25: 3514-3516). The recognition mechanisminvolved does not utilize the major histocompatability class (MHC) Iantigen presentation pathway and thus NK cells are neither antigen orMHC restricted and more importantly do not undergo clonal expansion tobe effective (Trinchieri, 1989, Adv. Immunology 47: 176-187). Inaddition to their cytotoxic actions, NK cells have the ability tomodulate the immune system by the production of plietropic cytokinesupon cellular activation.

The activation of NK cells largely depends on NK triggering receptors,NKG2D, CD16 and the recently identified natural cytoxicity receptors(NCR) (Arnon et al., 2006, Seminars in Cancer Biology 16: 348-358),which include three members: NKp46, NKp44 and NKp30 (Moretta et al.,2002, Scand. J. Immunol. 55: 229-232, Bottino et al., 2005, Trends inImmunology 26: 221-226). The NCR have recently been designated clusterof differentiation notation, with NKp44 designated CD336.

CD336 encodes a 44 kDa surface glycoprotein characterized by a proteinbackbone of approximately 29 kDa (Vitale et al., 1998, J. Exp. Med. 187:2065-2072). CD336 is not expressed on resting but only on activated NKcells, thus the surface display of CD336 can be used as a surrogatemarker of NK activation (Moretta et al., 2001, Annu. Rev. Immunol. 19:197-223). While CD336 is a pertinent marker of cell activation, thehallmark of NK functionality is the ability to lyse target cells,typically NK lytic activity is measured in vitro using a cell linedeficient for surface MHC I expression such as the K562 tumor cell line.

The present inventors have identified novel methods for inducing an NKcell-mediated immune response, for increasing the activity of NK cellsand for assessing and detecting an NK cell response in connection withthe treatment of viruses and tumors.

BRIEF SUMMARY OF THE INVENTION

In a first aspect, this invention provides a method for inducing acytotoxic NK cell-mediated immune response in a mammalian subject, whichcomprises the steps of isolating peripheral blood mononuclear cells(PBMCs) from a subject, exposing the PBMCs in vitro to a proteinconjugate comprising granulocyte macrophage colony stimulating factor(GM-CSF) covalently linked to a soluble peptide antigen selected fromthe group consisting of a tumor associated antigen (TAA) and an oncogeneproduct, under conditions effective to activate the PBMCs, wherein thePBMCs are effective in activating NK cells to produce a cytotoxiccellular response that is higher than that produced by the PBMCs whichhave not been activated by the protein conjugate, and administering theactivated PBMCs to the subject.

In another aspect, the invention provides a method for increasing NKcell activity, comprising the steps of isolating peripheral bloodmononuclear cells (PBMCs) from a subject, exposing the PBMCs in vitro toa protein conjugate comprising granulocyte macrophage colony stimulatingfactor (GM-CSF) covalently linked to a soluble peptide antigen selectedfrom the group consisting of a tumor associated antigen (TAA) and anoncogene product, under conditions effective to activate the PBMCs,wherein the PBMCs are effective in activating NK cells.

In yet another aspect, the invention provides an improvement fordetermining whether the individual subject is a candidate for additionaltreatment by administration of the activated PBMCs by assessing theresponse of an individual subject to an anti-cancer therapy comprisingthe steps of (a) isolating PBMCs from a subject; (b) exposing the PBMCsin vitro to a protein conjugate comprising granulocyte macrophage colonystimulating factor (GM-CSF) covalently linked to a soluble peptideantigen selected from the group consisting of a tumor associated antigen(TAA) and an oncogene product, under conditions effective to activatethe PBMCs; (c) administering the activated PBMCs to the subject; (d)repeating step (a) and (b) at least 10 days after previous step (c) hasoccurred, (e) assessing the NK activity of the activated PBMCs from thesecond isolation; and (f) if the NK activity has increased significantlyover the level of NK activity prior to the first administration,classifying the subject as a good candidate for additional treatment byactivated PBMC administration.

In another aspect, the invention provides a method for determiningwhether a subject has had a therapeutically effective response toadministration of activated PBMCs comprising the steps of (a) isolatingPBMCs from a subject; (b) exposing the PBMCs in vitro to a proteinconjugate comprising granulocyte macrophage colony stimulating factor(GM-CSF) covalently linked to a soluble peptide antigen selected fromthe group consisting of a tumor associated antigen (TAA) and an oncogeneproduct, under conditions effective to activate the PBMCs; (c)administering the activated PBMCs to the subject; (d) repeating step (a)and (b) at least 10 days after previous step (c) has occurred, (e)assessing the NK activity of the activated PBMCs from the previousisolation; and (f) determining the change in the NK activity over thelevel of NK activity of the activated PBMCs prior to the firstadministration.

The methods of the present invention are particularly suited to thetreatment of cancers such as, for example, soft tissue sarcomas,lymphomas, and cancers of the brain, esophagus, uterine cervix, bone,lung, endometrium, bladder, breast, larynx, colon/rectum, stomach,ovary, pancreas, adrenal gland and prostate. Exemplified herein aremethods for the treatment of prostate and/or breast cancer.

In each of the above aspects of the invention, (i) the PBMCs may beantigen presenting cells (APCs); (ii) PBMCs may be dendritic cells(DCs); (iii) for use in treating a tumor, the soluble peptide antigenmay be a TAA, including a tissue-specific tumor antigen; (iv) for use intreating a tumor, the soluble peptide antigen may be an oncogeneproduct; (v) the protein conjugate may further include a linker peptidejoining the GM-CSF to the soluble peptide antigen; (vi) for use intreating prostate cancer, the tissue-specific tumor antigen may beprostatic acid phosphatase (PAP) having at least 95% sequence identityto the sequence depicted as SEQ ID NO: 1; (vii) the protein conjugatemay be a fusion protein having at least 95% sequence identity with thesequence depicted as SEQ ID NO: 5; (vii) for use in treating breastcancer, the oncogene product may be Her2; (viii) the protein conjugatemay comprise a fusion protein having at least 95% sequence identity withthe sequence depicted as SEQ ID NO: 7; the mammalian subject is a human;and (ix) the protein conjugate may be produced in a baculovirusexpression system.

Also in aspects of the invention involving the steps of (a) isolatingPBMCs from a subject; (b) exposing the PBMCs in vitro to a proteinconjugate comprising granulocyte macrophage colony stimulating factor(GM-CSF) covalently linked to a soluble peptide antigen selected fromthe group consisting of a tumor associated antigen (TAA) and an oncogeneproduct, under conditions effective to activate the PBMCs, and (c)administering the activated PBMCs to the subject; the claimed inventionmay further comprise repeating steps (a), (b) and (c) at least once witheach cycle beginning at least ten days after step (c) has occurred; andsteps (a) through (c) may be performed a total of three times andwherein fourteen days has elapsed since the previous step (c) hasoccurred.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. FIG. 1 shows NK cell activity as measured by CD336 surfaceexpression on CD16+ and CD56+ NK cells before and after culture withsipuleucel-T, an investigative immunotherapeutic agent manufactured byDendreon Corp, Seattle, Wash., at weeks 0, 2 and 4. FIG. 1 shows thatCD336 surface expression on both CD16+ and CD56+ cells is enhancedpost-culture. Pre- and post culture cells were surface stained for CD16,CD56 and CD336, and 200,000 events were collected on a Becton DickinsonFACSAria flow cytometer. Gated CD16+ and CD56+ cells were then analyzedfor CD336 expression and the percent of CD16+ or CD56+ cells thatexpressed CD336 then calculated.

FIG. 2. FIG. 2 shows NK cell activity as measured by CD336 surfaceexpression on CD16+ and CD56+ NK cells from subjects that received aplacebo (cells incubated without the GM-CSF fusion protein) in thedouble-blind, placebo controlled clinical trial for sipuleucel-T. FIG. 2shows that CD336 surface expression on both CD16+ and CD56+ cells is notenhanced post-culture. Pre- and post culture cells were surface stainedfor CD16, CD56 and CD336, and 200,000 events were collected on a BectonDickinson FACSAria flow cytometer. Gated CD16+ and CD56+ cells were thenanalyzed for CD336 expression and the percent of CD16+ or CD56+ cellsthat expressed CD336 then calculated.

FIG. 3. FIG. 3 shows sipuleucel-T cell lytic activity against the MHC-Ideficient cell line K562 at weeks 0, 2 and 4 for several differentsubjects. FIG. 2 shows that sipuleucel-T cells generated from the week 2apheresis from seven different subjects that had received the week 0treatment, possessed cytotoxic activity as gauged by lysis of the K562tumor cell line. Sipuleucel-T cells were titrated in triplicate at aneffector to target ratio starting at 50:1 against a fixed number of K562target cells. The cells were incubated at 37° C. for 4 hours after whichtime the medium was tested in a colorimetric assay for the presence ofthe intracellular enzyme lactate dehydrogenase (LDH). The degree oflytic activity was thus calculated using the following formula:

${\%\mspace{14mu}{Cytotxicity}} = {100\; \times \frac{A - B - C}{D - C}}$

-   A=LDH from test cell mixture (effector cells+target cells)-   B=spontaneous LDH from effector cells-   C=spontaneous LDH from target cells-   D=maximal LDH from target cells

FIG. 4. FIG. 4 shows sipuleucel-T cell lytic activity against the MHC-Ideficient cell line K562 at weeks 0, 2 and 4 from two subjects thatreceived a placebo (cells incubated without the GM-CSF fusion protein)in the double bind, placebo cotrolled clinical trial for sipuleucel-T.FIG. 4 shows that cells generated from the week 2 apheresis from twosubjects who had received the week 0 placebo, did not possess cytotoxicactivity as gauged by lysis of the K562 tumor cell line. Sipuleucel-Tcells were titrated in triplicate at an effector to target ratiostarting at 50:1 against a fixed number of K562 target cells. The cellswere incubated at 37° C. for 4 hours after which time the medium wastested in a colorimetric assay for the presence of the intracellularenzyme lactate dehydrogenase (LDH). The degree of lytic activity wasthus calculated using the following formula:

${\%\mspace{14mu}{Cytotxicity}} = {100 \times \frac{A - B - C}{D - C}}$

-   A=LDH from test cell mixture (effector cells+target cells)-   B=spontaneous LDH from effector cells-   C=spontaneous LDH from target cells-   D=maximal LDH from target cells

FIG. 5. FIG. 5 is a diagrammatic representation of a portion of a 96well V-bottomed plate on which the major histocompatability complex(MHC) I negative cell line K562 (also referred to as “target cells”) andsipuleucel-T cells (also referred to as “effector cells”) were used toassess the NK lytic activity of sipuleucel-T.

SEQ ID NO: 1 is the amino acid sequence of human prostatic acidphosphatase (huPAP) as encoded by the cDNA sequence depicted in SEQ IDNO: 2.

SEQ ID NO: 2 is the nucleotide sequence of a cDNA encoding humanprostatic acid phosphatase (huPAP) as depicted in SEQ ID NO: 1.

SEQ ID NO: 3 is the amino acid sequence of a humangranulocyte-macrophage colony stimulating factor (huGM-CSF) as encodedby the cDNA sequence depicted in SEQ ID NO: 4.

SEQ ID NO: 4 is the nucleotide sequence of a cDNA encoding humangranulocyte-macrophage colony stimulating factor (huGM-CSF) as depictedin SEQ ID NO: 3.

SEQ ID NO: 5 is the amino acid sequence of a human prostatic acidphosphatase/human granulocyte-macrophage colony stimulating factor(huPAP/huGM-CSF) fusion protein as encoded by the cDNA sequence depictedin SEQ ID NO: 6,

SEQ ID NO: 6 is the nucleotide sequence of a cDNA encoding humanprostatic acid phosphatase/human granulocyte-macrophage colonystimulating factor (huPAP/huGM-CSF) fusion protein as depicted in SEQ IDNO: 5.

SEQ ID NO: 7 is the amino acid sequence of a HER500-humangranulocyte-macrophage colony stimulating factor (HER500-huGM-CSF)fusion protein as encoded by the cDNA sequence depicted in SEQ ID NO: 8.

SEQ ID NO: 8 is the nucleotide sequence of a cDNA encoding aHER500-human human granulocyte-macrophage colony stimulating factor(HER500-huGM-CSF) fusion protein as depicted in SEQ ID NO: 7.

DETAILED DESCRIPTION OF THE INVENTION

As described above, the invention provides a method for inducing acytotoxic NK cell-mediated immune response in a mammalian subject, whichcomprises the steps of isolating peripheral blood mononuclear cells(PBMCs) from a subject, exposing the PBMCs in vitro to a proteinconjugate comprising a GM-CSF having at least 95% sequence identity withthe sequence depicted in SEQ ID NO: 3 (GM-CSF), covalently linked to asoluble peptide antigen selected from the group consisting of a tumorassociated antigen and an oncogene product, under conditions effectiveto activate the PBMCs, wherein the PBMCs are effective in activating NKcells to produce a cytotoxic cellular response that is higher than thatproduced by the PBMCs when not activated by the protein conjugate, andadministering the activated PBMCs to the subject.

In another aspect, the invention provides a method for increasing NKcell activity, comprising the steps of isolating peripheral bloodmononuclear cells (PBMCs) from a subject, exposing the PBMCs in vitro toa protein conjugate comprising GM-CSF covalently linked to a solublepeptide antigen selected from the group consisting of a tumor associatedantigen (TAA) and an oncogene product, under conditions effective toactivate the PBMCs, wherein the PBMCs are effective in activating NKcells.

In yet another aspect, the invention provides an improvement fordetermining whether the individual subject is a candidate for additionaltreatment by administration of the activated PBMCs by assessing theresponse of an individual subject to an anti-cancer therapy comprisingthe steps of (a) isolating PBMCs from a subject; (b) exposing the PBMCsin vitro to a protein conjugate comprising GM-CSF covalently linked to asoluble peptide antigen selected from the group consisting of a tumorassociated antigen (TAA) and an oncogene product, under conditionseffective to activate the PBMCs; (c) administering the activated PBMCsto the subject; (d) repeating step (a) and (b) at least 10 days afterprevious step (c) has occurred, (e) assessing the NK activity of theactivated PBMCs from the second isolation; and (f) if the NK activityhas increased significantly over the level of NK activity prior to thefirst administration, classifying the subject as a good candidate foradditional treatment by activated PBMC administration.

In another aspect, the invention provides a method for determiningwhether a subject has had a therapeutically effective response toadministration of activated PBMCs comprising the steps of (a) isolatingPBMCs from a subject; (b) exposing the PBMCs in vitro to a proteinconjugate comprising GM-CSF covalently linked to a soluble peptideantigen selected from the group consisting of a tumor associated antigen(TAA) and an oncogene product, under conditions effective to activatethe PBMCs; (c) administering the activated PBMCs to the subject; (d)repeating step (a) and (b) at least 10 days after previous step (c) hasoccurred, (e) assessing the NK activity of the activated PBMCs from theprevious isolation; and (f) determining the change in the NK activityover the level of NK activity of the activated PBMCs prior to the firstadministration.

In each of the aspects of the invention, (i) the PBMCs may be antigenpresenting cells (APCs); (ii) PBMCs may be dendritic cells (DCs); (iii)for use in treating a tumor, the soluble peptide antigen may be a TAA,including a tissue-specific tumor antigen; (iv) for use in treating atumor, the soluble peptide antigen may be an oncogene product; (v) theprotein conjugate may further include a linker peptide joining theGM-CSF to the soluble peptide antigen; (vi) for use in treating prostatecancer, the tissue-specific tumor antigen may be prostatic acidphosphatase (PAP) having at least 95% sequence identity to the sequencedepicted as SEQ ID NO: 1; (vii) the protein conjugate may be a fusionprotein having at least 95% sequence identity with the sequence depictedas SEQ ID NO: 5; (vii) for use in treating breast cancer, the oncogeneproduct may be Her2; (viii) the protein conjugate may comprise a fusionprotein having at least 95% sequence identity with the sequence depictedas SEQ ID NO: 7; the mammalian subject is a human; and (ix) the proteinconjugate may be produced in a baculovirus expression system.

Immunotherapeutic Compositions

Within certain embodiments, the present invention provides that thePBMCs are isolated antigen presenting cells (APCs) obtained from asubject. In certain embodiments, the APCs are stimulated by exposure invitro to a tumor-associated antigen (TAA). The tumor-associated antigenmay be a tissue-specific tumor antigen. As used herein, Thetumor-associated antigen and/or the tissue-specific tumor antigen are acomponent of an immunotherapeutic composition that comprises a proteinconjugate wherein the protein conjugate comprises an N-terminal moietyand a C-terminal moiety, wherein the C-terminal moiety has at least 95%sequence identity with the sequence depicted as SEQ ID NO: 3 (huGM-CSFor GM-CSF). In certain preferred embodiments, the APCs are stimulatedwith a protein conjugate comprising an N-terminal moiety, having atleast 95% sequence identity with the sequence depicted in SEQ ID NO: 1(huPAP or PAP) or an active fragment, derivative, or variant of huPAP.In an especially preferred embodiment the subject's APCs are stimulatedby a protein conjugate comprising the sequence depicted in SEQ ID NO: 5(PAP/GM-CSF)

In other embodiments, the APCs are stimulated in vitro by exposure to aprotein conjugate comprising a C-terminal moiety comprising GM-CSF andan N-terminal moiety comprising an oncogene product. The oncogeneproduct is a component of an immunotherapeutic composition thatcomprises a protein conjugate wherein the protein conjugate comprises anN-terminal moiety and a C-terminal moiety, wherein the C-terminal moietyis GM-CSF. In a preferred embodiment, the N-terminal moiety having atleast 95% sequence identity with the sequence depicted in SEQ ID NO: 7(HER500-hGM-CSF). The immunotherapeutic compositions described hereinare effective in inducing an NK cell-mediated immune response againstthe protein conjugate. The NK cell-mediated immune response is higherthan that produced by APCs when not exposed to the protein conjugate.Specific preferred embodiments provide that the APCs are dendritic cells(DCs).

APCs and DCs

As used herein, the term “antigen presenting cells” or “APCs” refers tocells that are capable of inducing an NK cell-mediated immune response,and include, but are not limited to certain macrophages, B cells, and,most preferable, dendritic cells (DCs). “Dendritic cells” or “DCs” aremembers of a diverse population of morphologically similar cell typesfound in lymphoid or non-lymphoid tissues. These cells are characterizedby their distinctive morphology and high levels of surface MHC class IIexpression (Steinman et al., 1991, Ann. Rev. Immunol. 9: 271).

APCs and DCs may be isolated from a number of tissue sources, andconveniently from peripheral blood. APCs and DCs may be isolated byroutine methodologies that are readily available in the art. Anexemplary suitable methodology for isolation of DCs is disclosed in U.S.Pat. Nos. 5,976,546, 6,080,409, and 6,210,662, each of these patents isincorporated herein by reference. Briefly, buffy coat cells may beprepared from peripheral blood. Cells may be harvested from leukopacs,layered over columns of organosilanized colloidal silica (OCS)separation medium (prepared as described by Dorn in U.S. Pat. No.4,927,749, incorporated herein by reference) at a density 1.0770 g/ml,pH 7.4, 280 mOsm/kg H₂O) in centrifuge tubes or devices. The OCS mediumis preferable prepared by reacting and thus blocking the silanol groupsof colloidal silica (approximately 10-20 nm diameter particles) with analkyl tri-methoxy silane reagent.

In one embodiment, the OCS density gradient material is diluted to anappropriate specific density in a physiological salt solutionsupplemented with polyvinylpyrolidone (PVP). The tubes are centrifugedand the PBMCs present at the interface, are harvested.

PBMC are resuspended and centrifuged again to remove platelets and mayoptionally be spun through columns of OCS (density 1.0650 g/ml, 280mOsm/kg H₂O). The resulting interface and PBMCs are harvested and washedwith D-PBS by centrifugation. The pellet fraction is resuspended in cellculture medium and cultured with the protein conjugate in a humidified5% CO₂ incubator for approximately 40 hours. Following incubation, thecells are harvested.

In a preferred embodiment, sipuleucel-T, an investigativeimmunotherapeutic agent manufactured by Dendreon Corp, (Seattle, Wash.)is generated from a subject's own blood cells using an apheresis. Thesubject's apheresis cells are centrifuged to remove autologous plasma,they are then resuspended in 0.9% sodium chloride USP solution andpassed through a buoyant density solution (BDS) of 1.077 g/ml gravity.The interface cells are collected and washed in 0.9% sodium chloride USPsolution after which they are then passed over a BDS 1.065 g/ml gravityseparation solution. The cells that pass through the density solutionare then collected and washed in 0.9% sodium chloride USP solution.These cells, termed BDS65 cells are cultured in AIM-V® culture mediumfor up to 44 hours with PA2024, a fusion protein comprising humanprostatic acid phosphatase fused to human GM-CSF. The cultured cells arethen washed out of the culture medium and resuspended in lactatedringers solution and are re-infused back into the subject. This processis performed three times, with each cycle of apheresis and culture beingconducted two weeks apart.

Protein Conjugates

Preferred protein conjugates comprise an N-terminal moiety whichincludes at least a portion of a tumor associated antigen or an oncogeneproduct and a C-terminal moiety which includes the dendritic cellbinding protein, GM-CSF.

As used herein, the term “tumor-associated antigen” refers to an antigenthat is characteristic of a tissue type, including specific tumortissues. An example of a tumor-associated antigen expressed by a tumortissue is the antigen prostatic acid phosphatase (PAP), which is presenton over 90% of all prostate tumors. The term “tissue specific tumorantigen” can be characterized as (i) inclusive of antigens that arecommon to a specific type of tumor and (ii) exclusive of antigens thatare specific only to an individual tumor. The term “oncogene product”refers to any protein encoded by a gene associated with cellulartransformation. Examples of oncogene products include, for example,Her2, p21RAS, and p53.

The terms “antigen presenting cell binding protein” and “dendritic cellbinding protein” refer to any protein for which receptors are expressedon an APC or a DC, respectively. Examples of antigen presenting cellbinding proteins and dendritic cell binding proteins include, but arenot limited to, GM-CSF, IL-1, TNF, IL-4, CD40L, CTLA4, CD28, and FLT-3ligand.

“Protein conjugates,” as disclosed herein, refer to covalent complexesformed between the N-terminal moiety and the C-terminal moiety. Proteinconjugates between tumor associated antigens/tumor-specificantigens/oncogene products and antigen presenting cell bindingproteins/dendritic cell binding proteins may be complexed eitherchemically or as a fusion protein.

The exemplary PAP/GM-CSF protein conjugate disclosed herein waspreviously described within U.S. Pat. Nos. 5,976,546, 6,080,409, and6,210,662, each of which is incorporated herein by reference and ispresented herein as SEQ ID NO: 5. This protein conjugate is a fusionprotein between a 386 amino acid portion of PAP at the N-terminus and a127 amino acid portion of GM-CSF at the C-terminus. In addition, thePAP/GM-CSF fusion protein further comprises, between the N-terminalmoiety and the C-terminal moiety, a two amino acid peptide linker havingthe sequence gly-ser. The fusion protein is manufactured in aBaculovirus expression system using sf21 insect cells.

As described above, the PAP/GM-CSF protein conjugate is exposed to asubject's PBMCs under conditions effective to activate the PBMCs and theactivated PBMCs are administered to the subject to induce a cytotoxic NKcell-mediated immune response.

The term “administration” or “administering” refers to various methodsof contacting a substance with a mammal, especially a human. Modes ofadministration may include, but are not limited to, methods that involvecontacting the substance intravenously, intraperitoneally, intranasally,transdermally, topically, subcutaneously, parentally, intramuscularly,orally, or systemically, and via injection, ingestion, inhalation,implantation, or adsorption by any other means. One exemplary means ofadministration of the protein conjugates or fusion proteins of thisinvention is via intravenous delivery, where the protein conjugate orfusion protein can be formulated as an aqueous solution, a suspension,or an emulsion, etc. Other means for delivering the protein conjugatesor fusion proteins of this invention includes intradermal injection,subcutaneous injection, intramuscular injection or transdermalapplication as with a patch.

Another exemplary protein conjugate disclosed herein is theHER500-hGM-CSF fusion protein that was previously described within U.S.Pat. Nos. 5,976,546, 6,080,409, 6,210,662, and 7,060,279 each of whichis incorporated herein by reference and is presented herein as SEQ IDNO: 7. This protein conjugate is a fusion protein that is composed of289 amino acids from the N-terminal extra-cellular domain and 217 aminoacids from the C-terminal intra-cellular domain of Her2 fused to 127amino acids of human GM-CSF at the C-terminus. The fusion protein ismanufactured in a Baculovirus expression system using sf21 insect cells.

In a preferred embodiment, the invention provides a method of inducing acytotoxic NK cell-mediated immune response in a human subject comprisingthe steps of (a) isolating APCs from the subject; (b) exposing the APCsin vitro to a protein conjugate comprising GM-CSF covalently linked toPAP, under conditions effective to activate APCs; (c) administering theactivated APCs to the subject; and (d) repeating steps (a)-(c) at leastonce with each cycle beginning at least 10 days after step (c) hasoccurred. In an especially preferred embodiment, steps (a)-(c) arerepeated one time with step (a) occurring 14 days after step (c).

In another aspect, the invention provides a method for increasing NKcell activity in a patient, comprising the steps of (a) isolating APCsfrom the patient; (b) exposing the APCs in vitro to a protein conjugatecomprising GM-CSF covalently linked to PAP, under conditions effectiveto activate APCs; (c) administering the activated APCs to the patient;and (d) repeating steps (a)-(c) at least once with each cycle beginningat least 10 days after step (c) has occurred. In an especially preferredembodiment, steps (a)-(c) are repeated one time with step (a) occurring14 days after step (c).

In another aspect, the invention provides a method of detecting in asubject a cytotoxic NK cell-mediated immune response comprising thesteps of (a) isolating APCs from the subject; (b) exposing the APCs invitro to a protein conjugate comprising GM-CSF covalently linked to asoluble peptide antigen selected from the group consisting of atissue-specific tumor antigen and an oncogene product, under conditionseffective to activate the APCs; (c) administering the activated APCs tothe subject; (d) repeating steps (a) and (b); and (e) detecting an NKcell response in the activated PBMCs. In certain embodiments of theinvention, the NK cell response is detected in vitro by CD336 surfaceexpression. In other embodiments of the invention, the NK cell responseis detected in vitro by lysis of the K562 tumor line.

Evaluation of NK Cell Activation

In one embodiment of the invention, NK cell activation is evaluated byflow cytometry of CD336 surface expression. APCs are obtained fromsubjects as described above and evaluated before and after culture withthe protein conjugates described above. Pre- and post culture cells weresurface stained for CD16, CD56 and CD336, and data were collected on aBecton Dickinson FACSAria flow cytometer. Gated CD16+ and CD56+ cellsare then analyzed for CD336 expression and the percent of CD16+ or CD56+cells that expressed CD336 then calculated. The results, as shown inFIG. 1, show that NK cell activity is enhanced post-culture with aPAP/GM-CSF protein conjugate and subsequent administration to thesubject.

In another embodiment of the invention, NK cell activation is evaluatedby lysis of the K562 tumor cell line. The K562 cell line is widely usedas a target for NK activity as it is MHC class I negative and thereforecannot present either autologous or antigen derived peptides (Ortaldo etal., 1977, J. Natl. Cancer Inst. 59: 77-82). The details of material andmethods used for the NK lytic cell assay are described below in Example2. The results, as shown in FIG. 2, show that APCs generated from theweek 2 apheresis from subjects undergoing treatment with sipuleucel-Tpossessed cytotoxic activity as gauged by lysis of the K562 cell line.

EXAMPLES

The following examples are provided by way of illustration only and notby way of limitation. Those of skill in the art will readily recognize avariety of non-critical parameters that could be changed or modified toyield essentially similar results.

Example 1

Materials and Methods

PA2024 is a proprietary recombinant fusion protein containing PAP andGM-CSF sequences manufactured by Dendreon Corporation (Seattle, Wash.)for the investigational cellular immunotherapy sipuleucel-T. PA2024 isexpressed in a baculovirus system.

All subject and healthy donor specimens were collected according toinvestigator sponsored protocols approved by the appropriateInvestigational Review Board. After receiving informed consent, whiteblood cells were collected by apheresis and prepared for transportand/or processing. The subject's apheresis cells were centrifuged toremove autologous plasma, they are then resuspended in 0.9% sodiumchloride USP solution and passed through a buoyant density solution(BDS) of 1.077 g/ml gravity. The interface cells were collected andwashed in 0.9% sodium chloride USP solution after which they were thenpassed over a BDS 1.065 g/ml gravity separation solution. The cells thatpass through the density solution were then collected and washed in 0.9%sodium chloride USP solution. These cells, termed BDS65 cells werecultured in AIM-V® culture medium for up to 44 hours with PA2024, afusion protein comprising human prostatic acid phosphatase fused tohuman GM-CSF. The cultured cells were then washed out of the culturemedium and resuspended in lactated ringers solution and were re-infusedback into the subject. This process was performed three times, with eachcycle of apheresis and culture being conducted two weeks apart.

1×10⁷ pre-culture and post-culture cells were pelletted bycentrifugation and then resuspended in 1 ml of Dulbeccos PhosphateBuffered Saline (D-PBS) containing 10% normal mouse serum (NMS) andincubated at room temperature for 10 minutes. After this time the cellswere then centrifuged again and the supernatant was aspirated and thecells were resuspended in 1 ml of staining buffer (D-PBS containing 2%Bovine Serum Albumin—BSA). Aliquots of 1×10⁶ cells were then stainedwith the following combination of antibodies in the wells of a 96 wellround bottom staining plate: Fluoroisothiocyanate (FITC) labeled murineIgG1 together with Phycoerythrin (PE) labeled murine IgG1 andPhycoerythrin-Cyanate 5 (PE-Cy5) labeled murine IgG1, FITC labeledmurine anti-human CD16 together with PE labeled murine anti-human CD336and PE-Cy5 labeled murine anti-human CD56. The cells were incubated inthe dark at 4° C. for 20 minutes after which time 100 μl of stainingbuffer was added and the plate was centrifuged for 5 minutes, thesupernatant was aspirated off and the cells were then resuspended in atotal volume of 200 μl of D-PBS containing 1% parformaldehyde. Fixedcells were then acquired on a Becton Dickinson FACSAria where a total of200,000 gated events were collected. Flow data was then analyzed usingBeckman Coulter CXP software; the cells stained with FITC labeled murineIgG1, PE labeled murine IgG1 and PECy5 labeled murine IgG1 were used toestablish non specific staining. To define the CD16+ and CD56+ NK cellpopulations, the signals for these surface markers had to be greaterthan the signal from the FITC or PECy5 labeled murine IgG1 stainedcells. Gated CD16+ and CD56+ NK cells were then assessed for CD336staining, with positive staining for CD336 being a signal that wasgreater than that detected by staining with PE labeled murine IgG1 andthe number of CD16+ or CD56+ cells positive for CD336 were expressed asa percentage of the CD16+ or CD56+ NK cell populations.

Results

The results, as presented in FIG. 1, show that CD336 surface expressionon both CD16+ and CD56+ cells is enhanced post-culture withsipuleucel-T. The results, as presented in FIG. 2, also show that CD336expression is not enhanced for the subjects that did not receivesipuleucel-T.

Example 2

Materials and Methods

PA2024 is a proprietary recombinant fusion protein containing PAP andGM-CSF sequences manufactured by Dendreon Corporation (Seattle, Wash.)for the investigational cellular immunotherapy sipuleucel-T. PA2024 isexpressed in a baculovirus system.

All subject and healthy donor specimens were collected according toinvestigator sponsored protocols approved by the appropriateInvestigational Review Board. After receiving informed consent, whiteblood cells were collected by apheresis and prepared for transportand/or processing. The subject's apheresis cells were centrifuged toremove autologous plasma, they are then resuspended in 0.9% sodiumchloride USP solution and passed through a buoyant density solution(BDS) of 1.077 g/ml gravity. The interface cells were collected andwashed in 0.9% sodium chloride USP solution after which they were thenpassed over a BDS 1.065 g/ml gravity separation solution. The cells thatpass through the density solution were then collected and washed in 0.9%sodium chloride USP solution. These cells, termed BDS65 cells werecultured in AIM-V® culture medium for up to 44 hours with PA2024, afusion protein comprising human prostatic acid phosphatase fused tohuman GM-CSF. The cultured cells were then washed out of the culturemedium and resuspended in lactated ringers solution and were re-infusedback into the subject. This process was performed three times, with eachcycle of apheresis and culture being conducted two weeks apart.

NK lytic activity of sipuleucel-T was determined by assessing the degreeof lysis of the major histocompatability complex (MHC) I negative cellline K562, also referred to as target cells, by use of a Non-RadioactiveCytotoxicity Assay (Promega Cat#G1780, Instructions Part #TB163). K562cells were maintained in log phase culture in standard RPMI1640 mediasupplemented with 10% Fetal Bovine Serum (FBS) and on the day of usewere washed out of the RPMI/10% FBS medium by centrifugation. K562 cellswere then resuspended in RPMI1640 medium supplemented with 5% HumanSerum (HS) at a concentration of 2.5×10⁵/ml. 15×10⁶ sipuleucel-T cells,also referred to as effector cells, were washed and also resuspended in600 μl of RPMI/5% HS and 100 μl of the effector cells were dispensed intriplicate into the wells of a 96 well V-bottomed plate, in the firstcolumn of the Experimental and Effectors only section of the plate, asdetailed in FIG. 5.

The first column of cells represents the highest effector: target ratio,100 μl of RPMI/5% HS was then dispensed into every triplicate set ofwells of the Experimental, Effectors only, T_(max), (Target maximalrelease), T_(Spont)(Target spontaneous release), and media sets. Theplate was then centrifuged and the Effector cells were then seriallydiluted down the Experimental and Effectors only by transferring 100 μlof volume across the plate of the two aforementioned sets. 2.5×10⁴target cells were then dispensed in triplicate into the wells of theExperimental, T_(Spont) and T_(Max). sets and a further 100 μl ofRPMI/5% HS was added to the Media wells. The plate was then incubatedfor 3.5 hours at 37° C., 5% CO₂ after which time 20 μl of 10× lysisbuffer was added to the T_(Max) wells and the plate was then incubatedfor a further 30 minutes. The plate was then centrifuged for 4 minutesat 250G. 50 μl of supernatant was then transferred from each well to a96 well black walled flat bottomed plate and an equal volume ofsubstrate buffer was added to each well and the plate incubated at roomtemperature for 30 minutes. After this time 50 μl of stop solution wasadded and the optical density of each well was determined on an ELISAplate reader at a wavelength of 490 nm. The degree of lytic activity wasthe calculated using the following formula:

${\%\mspace{14mu}{Cytotxicity}} = \frac{{Experimental} - {{Effector}\mspace{14mu}{Spontaneous}} - {{Target}\mspace{14mu}{Spontaneous}}}{\begin{matrix}{{{Target}\mspace{14mu}{Maximum}} -} \\{{Target}\mspace{14mu}{Spontaneous}}\end{matrix}}$Results

The results, as presented in FIG. 3, show that sipuleucel-T cellsgenerated from the week 2 apheresis of treated subjects possessedcytotoxic activity as gauged by lysis of the K562 tumor cell line. Theresults, as presented in FIG. 4, also show that NK lytic activity is notgenerated at week 2 in the placebo subjects.

All patents, patent applications, and other publications cited in thisapplication, including published amino acid or polynucleotide sequences,are incorporated by reference in the entirety for all purposes.

What is claimed is:
 1. A method for detecting a cytotoxic NKcell-mediated immune response in a subject, comprising: (a) isolatingperipheral blood mononuclear cells (PBMCs) comprising dendritic cellsfrom the subject; (b) exposing said PBMCs in vitro to a proteinconjugate comprising granulocyte macrophage colony stimulating factor(GM-CSF) covalently linked to a soluble peptide antigen, said proteinconjugate comprising SEQ ID NO: 5 or SEQ ID NO: 7, under conditionseffective to activate said PBMCs; (c) assessing the level of an NK cellresponse in the activated PBMCs; (d) administering said activated PBMCsto the subject; (e) repeating steps (a) and (b) at least 10 days afterthe previous step (d) has occurred, resulting in a second isolation ofPBMCs; and (f) assessing the level of an NK cell response in theactivated PBMCs from the second isolation, wherein said activated PBMCsare effective in activating NK cells to produce a cytotoxic cellularresponse at a level higher than that produced by the PBMCs which havenot been activated by the protein conjugate.
 2. The method of claim 1,wherein said protein conjugate comprises a fusion protein having thesequence depicted as SEQ ID NO:
 5. 3. The method of claim 1, whereinsaid protein conjugate comprises a fusion protein having the sequencedepicted as SEQ ID NO:
 7. 4. The method of claim 1, wherein saidmammalian subject is a human.
 5. The method of claim 1, wherein saidprotein conjugate is produced in a baculovirus expression system.
 6. Themethod of claim 1, wherein said method further comprises repeating steps(a), (b) and (c) at least once with each cycle beginning at least 14days after step (c) has occurred.
 7. The method of claim 6, wherein saidstep (d) is performed in vitro by CD336 surface expression.
 8. Themethod of claim 6, wherein said step (d) is performed in vitro lysis ofthe K562 tumor line.
 9. A method for determining whether a subject hashad a therapeutically effective response to a protein conjugate,comprising, (a) isolating PBMCs comprising dendritic cells from thesubject; (b) exposing said PBMCs in vitro to a protein conjugatecomprising granulocyte macrophage colony stimulating factor (GM-CSF)covalently linked to a soluble peptide antigen, said protein conjugatecomprising SEQ ID NO: 5 or SEQ ID NO: 7, under conditions effective toactivate said PBMCs; (c) administering said activated PBMCs to thesubject; (d) repeating steps (a) and (b) at least 10 days after step (c)has occurred; and (e) determining a change in NK cell activity of theactivated PBMCs.
 10. The method of claim 9, wherein determining step (e)includes assessing the NK activity of the activated PBMCs from theprevious isolation; and determining the change in the NK activity overthe level of NK activity of the activated PBMCs prior to the firstadministration.
 11. The method of claim 9, wherein said step (e) isperformed in vitro by CD336 surface expression.
 12. The method of claim9, wherein said step (e) is performed in vitro lysis of the K562 tumorline.
 13. The method of claim 12, wherein said steps (a) through (c) areperformed a total of two times and wherein 14 days has elapsed since theprevious step (c) has occurred.
 14. The method of claim 9, wherein saidsteps (a) through (c) are performed a total of three times and wherein14 days has elapsed since the previous step (c) has occurred.